Nucleotide excision repair eliminates unique DNA-protein cross-links from mammalian cells
Nucleotide excision repair eliminates unique DNA-protein cross-links from mammalian cells
复制标题
DOI：
10.1074/jbc.m702856200
复制
发表时间：
2007-08-03
影响因子：
4.8
通讯作者：
O'Connor, Timothy R.
中科院分区：
文献类型：
--
作者：
Baker, David J.;Wuenschell, Gerald;O'Connor, Timothy R.
DNA-蛋白质交联（DPC）对细胞过程提出了一个可怕的障碍，因为它们与大多数化学加合物相比是“超级庞大”的。DPC的消除对细胞存活至关重要，因为它们的持续存在可能导致细胞死亡或通过阻碍DNA和RNA合成而停止细胞周期进程。为了研究DPC修复，我们使用DNA甲基转移酶在寡脱氧核糖核苷酸或质粒中产生独特的DPC加合物，以监测体外切除和体内修复。我们表明，HhaI DNA甲基转移酶共价结合到一个寡脱氧核糖核苷酸没有有效地切除，通过使用哺乳动物细胞的无提取物，但蛋白酶消化的全长HhaI DNA甲基转移酶-DPC产生的底物，有效地去除了类似的核苷酸切除修复（NER）的过程。为了检查该独特DPC的修复，我们开发了两种基于质粒的DPC修复体内测定法。一项试验表明，在非转录区，DPC修复在6小时内大于60%。另一种基于宿主细胞再活化的试验使用绿色荧光蛋白，证明转录基因中的DPC也被修复。使用Xpg缺陷型细胞（NER缺陷型）与体内宿主细胞再活化试验和独特的DPC表明，NER在修复该加合物中具有作用。我们还证明了26 S蛋白酶体在DPC修复中的作用。这些数据与修复模型一致，其中DPC的多肽链首先通过蛋白水解在NER之前减少。
DNA- protein cross-links (DPCs) present a formidable obstacle to cellular processes because they are "superbulky" compared with the majority of chemical adducts. Elimination of DPCs is critical for cell survival because their persistence can lead to cell death or halt cell cycle progression by impeding DNA and RNA synthesis. To study DPC repair, we have used DNA methyltransferases to generate unique DPC adducts in oligodeoxyribonucleotides or plasmids to monitor both in vitro excision and in vivo repair. We show that HhaI DNA methyltransferase covalently bound to an oligodeoxyribonucleotide is not efficiently excised by using mammalian cell-free extracts, but protease digestion of the full-length HhaI DNA methyltransferase-DPC yields a substrate that is efficiently removed by a process similar to nucleotide excision repair (NER). To examine the repair of that unique DPC, we have developed two plasmid-based in vivo assays for DPC repair. One assay shows that in nontranscribed regions, DPC repair is greater than 60% in 6 h. The other assay based on host cell reactivation using a green fluorescent protein demonstrates that DPCs in transcribed genes are also repaired. Using Xpg-deficient cells (NER-defective) with the in vivo host cell reactivation assay and a unique DPC indicates that NER has a role in the repair of this adduct. We also demonstrate a role for the 26 S proteasome in DPC repair. These data are consistent with a model for repair in which the polypeptide chain of a DPC is first reduced by proteolysis prior to NER.